DNase Protocol

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==Overview==
==Overview==
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==Materials==
==Materials==
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*The DNAfree kit contains the stuff you need: [https://products.appliedbiosystems.com/ab/en/US/adirect/ab;jsessionid=2LdHJLWLKLylLhcGty6LnMnXzP2qcg66GJJ0XTL7s2zKXPFyqwBN!1659201712?cmd=catNavigate2&catID=603181 Applied Biosystems]
*THAWED 10X DNase Buffer
*THAWED 10X DNase Buffer
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*
 
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*X μL reagent 2
 
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**component A (reagent 2 is made up of multiple components)
 
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**component B
 
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*equipment 1
 
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*equipment 2
 
==Procedure==
==Procedure==
#In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
#In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
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::*10ul 10X DNase buffer
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::*10μL 10X DNase buffer
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::*78ul dH<sub>2</sub>0
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::*78&mu;L dH<sub>2</sub>0
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::*2ul DNase
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::*2&mu;L DNase
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::*10ul RNA(1ug/ul)
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::*10&mu;L RNA(1ug/&mu;L)
#Incubate at 37&deg; C for 20-30 min.
#Incubate at 37&deg; C for 20-30 min.
#Add 0.1 volume resuspended DNase Inactivation Reagent.
#Add 0.1 volume resuspended DNase Inactivation Reagent.
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==Notes==
==Notes==
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#List troubleshooting tips here.   
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*[[User:Smoore|Smoore]] 09:59, 3 December 2008 (EST)DNase I requires calcium as well as magnesiumIf you are adding DNase I to degrade DNA in an existing solution (like a lysate), having 0.5-1.0 mM CaCl2 seems to really help.  I don't know if it is in the kit buffer.
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#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
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#Anecdotal observations that might be of use to others can also be posted here.   
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*[[User:Smoore|Smoore]] 09:59, 3 December 2008 (EST)If you are poor and rely on heating your samples to kill the DNase, make sure to chelate the metals with excess EDTA before heatingHot + metal = degraded RNA.
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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*Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
==References==
==References==

Current revision

back to protocols

Contents

Overview

Protocol for DNase treatment of total RNA. This uses a DNase inactivating reagent and thus avoids the DNase heat inactivation step that makes me nervous.

Materials

Procedure

  1. In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
  • 10μL 10X DNase buffer
  • 78μL dH20
  • 2μL DNase
  • 10μL RNA(1ug/μL)
  1. Incubate at 37° C for 20-30 min.
  2. Add 0.1 volume resuspended DNase Inactivation Reagent.
  3. Incubate 2 min at RT mixing every 30s.
  4. Centrifuge at 10,000xg for 1.5 min.
  5. Transfer solution to a new tube avoiding pelleted Inactivation Reagent.

Notes

  • Smoore 09:59, 3 December 2008 (EST)DNase I requires calcium as well as magnesium. If you are adding DNase I to degrade DNA in an existing solution (like a lysate), having 0.5-1.0 mM CaCl2 seems to really help. I don't know if it is in the kit buffer.
  • Smoore 09:59, 3 December 2008 (EST)If you are poor and rely on heating your samples to kill the DNase, make sure to chelate the metals with excess EDTA before heating. Hot + metal = degraded RNA.
  • Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

  1. Goldbeter A and Koshland DE Jr. . pmid:6947258. PubMed HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. . pmid:13718526. PubMed HubMed [Jacob-JMB-1961]
  3. Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164. [Ptashne-Genetic-Switch]
All Medline abstracts: PubMed HubMed

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


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